A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.
In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.
In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.
This is a list of topics in molecular biology. See also index of biochemistry articles.
In molecular biology, a hybridization probe(HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured into single stranded DNA (ssDNA) and then hybridized to the target ssDNA or RNA immobilized on a membrane or in situ.
Metagenomics is the study of genetic material recovered directly from environmental or clinical samples. The broad field may also be referred to as environmental genomics, ecogenomics, community genomics or microbiomics.
Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA, and proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pair sequence. DGGE was the original technique, and TGGE a refinement of it.
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
Terminal restriction fragment length polymorphism is a molecular biology technique for profiling of microbial communities based on the position of a restriction site closest to a labelled end of an amplified gene. The method is based on digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer. The result is a graph image where the x-axis represents the sizes of the fragment and the y-axis represents their fluorescence intensity.
Pseudomonas citronellolis is a Gram-negative, bacillus bacterium that is used to study the mechanisms of pyruvate carboxylase. It was first isolated from forest soil, under pine trees, in northern Virginia, United States.
Intrinsic, or rho-independent termination, is a process in prokaryotes to signal the end of transcription and release the newly constructed RNA molecule. In prokaryotes such as E. coli, transcription is terminated either by a rho-dependent process or rho-independent process. In the Rho-dependent process, the rho-protein locates and binds the signal sequence in the mRNA and signals for cleavage. Contrarily, intrinsic termination does not require a special protein to signal for termination and is controlled by the specific sequences of RNA. When the termination process begins, the transcribed mRNA forms a stable secondary structure hairpin loop, also known as a Stem-loop. This RNA hairpin is followed by multiple uracil nucleotides. The bonds between uracil and adenine are very weak. A protein bound to RNA polymerase (nusA) binds to the stem-loop structure tightly enough to cause the polymerase to temporarily stall. This pausing of the polymerase coincides with transcription of the poly-uracil sequence. The weak adenine-uracil bonds lower the energy of destabilization for the RNA-DNA duplex, allowing it to unwind and dissociate from the RNA polymerase. Overall, the modified RNA structure is what terminates transcription.
16S ribosomal RNA is the RNA component of the 30S subunit of a prokaryotic ribosome. It binds to the Shine-Dalgarno sequence and provides most of the SSU structure.
SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a next-generation DNA sequencing technology developed by Life Technologies and has been commercially available since 2006. This next generation technology generates 108 - 109 small sequence reads at one time. It uses 2 base encoding to decode the raw data generated by the sequencing platform into sequence data.
The versatility of polymerase chain reaction (PCR) has led to a large number of variants of PCR.
Saccharibacteria, formerly known as TM7, is a major bacterial lineage. It was discovered through 16S rRNA sequencing.
The Earth Microbiome Project (EMP) is an initiative founded by Janet Jansson, Jack Gilbert and Rob Knight in 2010 to collect natural samples and to analyze the microbial community around the globe.
Community fingerprinting is a set of molecular biology techniques that can be used to quickly profile the diversity of a microbial community. Rather than directly identifying or counting individual cells in an environmental sample, these techniques show how many variants of a gene are present. In general, it is assumed that each different gene variant represents a different type of microbe. Community fingerprinting is used by microbiologists studying a variety of microbial systems to measure biodiversity or track changes in community structure over time. The method analyzes environmental samples by assaying genomic DNA. This approach offers an alternative to microbial culturing, which is important because most microbes cannot be cultured in the laboratory. Community fingerprinting does not result in identification of individual microbe species; instead, it presents an overall picture of a microbial community. These methods are now largely being replaced by high throughput sequencing, such as targeted microbiome analysis and metagenomics.
Single-cell sequencing examines the sequence information from individual cells with optimized next-generation sequencing technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. For example, in cancer, sequencing the DNA of individual cells can give information about mutations carried by small populations of cells. In development, sequencing the RNAs expressed by individual cells can give insight into the existence and behavior of different cell types. In microbial systems, a population of the same species can appear genetically clonal. Still, single-cell sequencing of RNA or epigenetic modifications can reveal cell-to-cell variability that may help populations rapidly adapt to survive in changing environments.
Microbial DNA barcoding is the use of DNA metabarcoding to characterize a mixture of microorganisms. DNA metabarcoding is a method of DNA barcoding that uses universal genetic markers to identify DNA of a mixture of organisms.
